This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Lyme disease is caused by the tick-transmitted spirochete Borrelia burgdorferi (Bb). In the United States, a small percentage of patients with Lyme arthritis have persistent arthritis for months or even several years after treatment with 2-3 months of oral and/or IV antibotic therapy, termed antibiotic-refractory Lyme arthritis. In these patients, the arthritis persists despite negative results for B. burgdorferi DNA in joint fluid and synovial tissue in the post-antibiotic period. This work is focused on identification of a Bb glycolipid that induces an immune response in patients with Lyme disease, but the response disappears predictably after spirochetal killing. Towards this goal, we are extracting and fractionating Bb lipids, and testing individual fractions using ELISA assays with sera from Lyme disease patients or control sera from uninfected donors. Normal phase chromatography is employed for the initial lipid fractionation. This separates the lipids by (headgroup) class. Those fractions that are ELISA-positive are sub-fractionated by reversed-phase chromatography, which separates lipids by their hydrophobic moieties. These sub-fractions are again be tested by ELISA. Guided by serum reactivity, the complex lipid extract is being fractionated, so that off-line mass spectrometry can be used to identify the lipid structures, including that of the immunogenic component. We have extracted total lipids from Bb and separated them by normal phase chromatography. We have analyzed lipid fractions by MALDI-TOF and ESI qoTOF mass spectrometry to assess their complexity and to identify intact lipids for subsequent tandem MS structural identification. This approach has identified, by intact mass, several known Bb lipids, which will be used to identify possible immunogenic lipids or lipoproteins of the spirochete. We have also performed serum ELISA assays on acylated cholesteryl galactoside (ACG) purified from lipid fractions, an immunogenic glycolipid of Bb. Preliminary analysis shows that this lipid is immunogenic, and we are now working on the development of a reliable ELISA to assess the antibody response to this antigen. A manuscript describing the results from the lipid analyses has been accepted for publication in Clinical Immunology (Epub Apr 2, 2009).